Influenza is a major public health problem, with tens of thousands of deaths annually in the U.S. alone. Influenza viruses change due to mutations and to reassortment of viral genes, so new strains arise frequently. Current vaccines protect only against closely related strains, and thus are made from strains predicted to circulate during the coming season. If an unexpected strain of influenza or even a pandemic emerges, such vaccines may take too long to develop and prepare, so other immunization strategies should be explored. This lab studies ways to generate immunity protective against a wide range of influenza strains. Mouse studies have demonstrated potent, long-lasting immunity to multiple influenza A subtypes after exposure to virus of one subtype. We study the immune mechanisms responsible for this protection and the contributions to immunity of various viral components. Animals are immunized with whole virus, with plasmid DNA expressing individual influenza proteins, or with recombinant viral vectors of various types. Projects include studies of the role of IgA and other antibodies, and of cellular immunity mediated by T lymphocytes. More complete understanding of the immune responses mediating broad cross-protection would help us achieve safe and effective vaccination, and would suggest endpoints to monitor in preclinical and clinical trials of new vaccines. These studies with viral and plasmid vectors also provide information relevant to gene therapy using viral and plasmid DNA vectors. Outcomes in gene therapy are also affected by the immune responses induced by vector administration. We study the mouse influenza system with special focus on heterosubtypic immunity, that is, immunity induced by influenza A virus of one subtype (or its components) that provides cross-protection against challenge with influenza A virus of a different subtype. Vaccine development and prevention of pandemics would be aided by a more complete understanding of the broad cross-protection against widely divergent viral strains that is observed in animals. Main projects: a) Immune responses induced by DNA vaccination: Plasmid DNA vaccines encoding conserved influenza virus antigens NP and M reduce challenge virus replication and the resulting morbidity and mortality. We have prepared and tested constructs encoding other conserved influenza virus components. Some plasmids also have features intended to increase expression of the inserted genes and thus induce more potent immunity. We are currently using these constructs along with recombinant viruses in a variety of prime and boost regimens, to investigate protection against challenge and also patterns of epitope dominance. To our previous techniques of antibody analysis and cytotoxic T lymphocyte analysis, we have added intracytoplasmic staining for interferon-gamma and TNF-alpha produced by CD4+ or CD8+ T cells, and ELISPOT for lymphocytes producing IFN-gamma, IL-2, and IL-4. We have identified acidic polymerase (PA) as an additional conserved component that induces immunity protective against lethal challenge. The mechanism of this protection is under study. b) Potential pandemic subtypes: We previously showed that vaccination with DNA encoding the conserved antigens NP and M of influenza virus A/PR/8 (H1N1) protected against lethal challenge with some H5N1 viruses from the 1997 outbreak in Hong Kong. In the absence of an antigenically-matched HA-based vaccine, this approach may be a useful first line of defense against a rapidly spreading pandemic virus. We are now developing additional vaccination regimens that will be tested for ability to protect against challenge with novel influenza A subtypes (H5 and H9 planned). c) Role of IgA: The role of local, mucosal immune responses in protection against influenza, and in particular in heterosubtypic immunity, is incompletely understood. We have previously performed mucosal immunizations and heterosubtypic challenges in knockout mice lacking IgA, to analyze the role of this specialized mucosal antibody. Results showed these mice were capable of responses providing some cross-protection of both the upper and lower respiratory tract. However, under some conditions their handling of a primary infection and their protective immunity against heterosubtypic virus differed from that of wild type mice. In order to perform quantitative studies, we are backcrossing the IgA knockout mice with BALB/c, with monitoring by the MaxBax technology of Charles River. d) In collaboration with the lab of Dr. Gary Nabel, we are investigating influenza vaccination by a prime-and-boost strategy using recombinant adenoviral vectors. Antibody and T cell responses have been analyzed. Protection against high dose influenza challenge was seen, and required both the DNA priming and the recombinant adenovirus boosting. The scope of protection provided by this vaccine regimen is being further explored. e) Human heterosubtypic immunity: The possible impact of heterosubtypic immunity in humans was investigated by study of archival data from the Cleveland family study performed in the 1940's and 1950's. The 1957 pandemic with its shift from H1N1 to H2N2 virus provided a unique opportunity to analyze heterosubtypic immunity, because the entire human population was naive to H2 and N2 antigens. With IRB approval, records from that study were examined in Cleveland. The data suggest that adults were protected by a form of immunity children did not have. Detailed data is being analyzed.